Figure 5
NFAT5 regulation of intestinal cell differentiation. (a) Knockdown of NFAT5 attenuated NaBT-induced IAP and sucrase activities in Caco-2 cells. Caco-2 cells were transfected with control siRNA or siRNA targeting NFAT5. After a 24-h incubation, transfected cells were treated with NaBT (5 mM) for additional 72 h. Cells were lysed and alkaline phosphatase and sucrase activities were determined. (Data represent mean±S.D.; *P<0.05 versus control siRNA; #P<0.01 versus NaBT plus control siRNA, as determined by analysis of variance). (b) Overexpression of NFAT5 increased alkaline phosphatase promoter activity in Caco-2 cells. Caco-2 cells were co-transfected with a plasmid containing the intestinal alkaline phosphatase promoter fragment linked to the luciferase reporter gene and Myc-NFAT5 or empty vector. After a 48-h incubation, the transfected cells were harvested and luciferase activity was measured in the crude cell lysates, as described in Materials and Methods. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). (Data represent mean±S.D.; *P<0.01 versus control). (c) Knockdown of NFAT5 attenuated NaBT-induced IAP activity in HT29 cells. HT29 cells were transfected with control siRNA or siRNA targeting NFAT5. After a 24-h incubation, transfected cells were treated with NaBT (5 mM) for additional 24 h. Cells were lysed and alkaline phosphatase activity was determined. (Data represent mean±S.D.; *P<0.05 versus control siRNA; #P<0.01 versus NaBT plus control siRNA, as determined by analysis of variance).(d and e) Immunohistochemical analysis of NFAT5 protein expression in normal human colon (d) and small intestine (e). Tissue sections were fixed and stained with primary anti-human NFAT5 antibody. NFAT5 is mainly expressed in the differentiated region (that is, upper crypts and villus; arrows)
